PROJECT SUMMARY To adopt forms optimized for their functions, individual cells sometimes project remarkably elaborate membrane protrusions, and even arrange them in complex patterns on their surfaces. To create and support membrane structures, the underlying cortical cytoskeleton must be arranged in specific conformations. The particular complement of cytoskeletal associated proteins, and the local regulation of their activity, thus determines the shape and pattern of cell membrane protrusions. To understand how cytoskeletal proteins together create unique cellular structures, we will study the formation of stunning actin-based structures on the surface of zebrafish skin cells called microridges. Microridges (or similar structures called microplicae) are found on most mucosal epithelial cells, which not only form the outer layer of fish skin, but also many of our own tissues, including the cornea, mouth, and parts of our gut. The microridge-covered surfaces of these cells display a glycoprotein calyx and adsorb mucins, suggesting that the unique structure of microridges is optimized for mucus retention. Mucus protects vulnerable epithelial tissues from abrasion and drying out, so understanding how microridges form could provide insight into the etiology of diseases affecting mucosal tissues, such as dry eye and dry mouth conditions. This proposal builds on successful descriptive and discovery-based studies supported by an NIH R21 grant that led to the identification of several new proteins in microridges. The experiments proposed here investigate mechanisms by which these specific proteins contribute to microridge morphogenesis, and, from a broader perspective, how they function as an ensemble to create the unique shapes and properties of microridges. In Aim 1 we will test the hypothesis that two proteins, Ezrin and Drebrin-like, initiate the first step of microridge morphogenesis, the formation of microvilli-like microridge precursors. Aim 2 investigates the interactions between F-actin and intermediate filaments (IFs) in microridges by testing if F-actin patterning determines IF patterning, and by testing the hypothesis that two candidate proteins, Envoplakin and Periplakin, link these cytoskeletal elements together. Finally, in Aim 3, live imaging, pharmacology, and molecular approaches will be used to characterize how myosin-based contraction and Rho GTPase signaling contribute to microridge morphogenesis. Collectively these studies will provide mechanistic insights into microridge morphogenesis, illuminate how cytoskeletal proteins together create elaborate cellular structures, and potentially point to how defects in epithelial morphogenesis contribute to diseases afflicting mucosal epithelia.